Cotton fibers are usually produced by cultivating cotton plants of the genus Gossypium and collecting the cotton fibers from the capsules (cotton bolls) formed on the cotton plants. There are many varieties of cotton plant, from which cotton fibers with different fiber characteristics can be obtained and used for various applications depending on their fiber characteristics. Cotton fibers are characterized by various properties among which fiber length, fiber fineness and fiber strength are particularly important. Many efforts have been made so far to improve the characteristics of cotton fibers. Attempted improvements have been mainly focused on fiber length and fiber fineness. In particular, there has been a great demand for longer and finer cotton fibers. The variety of cotton plant known as Sea Island is famous for desired fiber characteristics; however, this variety of cotton plant exhibits a poor yield of cotton fibers and therefore the price of Sea Island cotton fibers is very high. If highly yielding cotton plants with cotton fiber characteristics equal to or better than those of Sea Island cotton can be produced, it will be a great contribution to industry.
The methods for improving the characteristics or yield of cotton fibers can be roughly classified into the following three categories:
1. Variety Improvement by Cross Breeding
This method has been utilized most widely so far. At present, almost all the cultivated varieties of cotton plant are bred by this method. However, much time is needed for this method and the degree of variability is limited, so that one cannot expect remarkable improvements in the cotton fiber characteristics or in the yield of cotton fibers.
2. Treatment with Plant Hormone
Plant hormones such as auxin, gibberellin, cytokinin and ethylene have been put to practical use widely for field crops or horticultural products. The influence of plant hormones on the fiber characteristics of cotton plants, particularly on the fiber elongation mechanism, is known by gibberellin, auxin or brassinolide; however, no effect has been fully confirmed yet, and it cannot be said that these plant hormones are effective for practical use.
3. Variety Improvement by Gene Recombination Technique
In recent years, gene recombinant technique has made remarkable progress, and several reports have been made on the successful variety improvement in certain kinds of plants (e.g., cotton, soybean, corn, tomato) by introduction and expression of a particular gene in these plants to confer a desired genetic trait thereon. There have been developed and put to practical use, for example, cotton plants with improved insect resistance by introduction of a gene coding for BT toxin (i.e., insecticidal protein toxin produced by Bacillus thuringiensis) or cotton plants with improved herbicide (Glyphosate) resistance by introduction of a gene coding for 5-enol-pyruvil-shikimic acid 3-phosphate synthetase.
If a gene associated with cotton fiber formation and elongation can be introduced into cotton plants and expressed in sufficient quantities, it would become possible to make a remarkable improvement in the characteristics or yield of cotton fibers. Further, the introduction of such a gene in anti-sense for makes it possible to suppress the action of this gene. In other words, it is believed that the characteristics and yield of cotton fibers can be controlled by introducing a gene associated with fiber formation and elongation into cotton plants, followed by large-scale expression or suppression of the gene. The method using such a genetic engineering technique can be expected to find wide applications, for example, more reliable control of fiber elongation and formation as compared with the conventional plant breeding by cross-breeding and screening. For this purpose, it is required that the mechanisms of fiber elongation and formation are elucidated on the genetic level and some genes closely associated with these mechanisms are discovered and then actually expressed and regulated in the cotton fiber tissues to control the mechanisms of fiber elongation and formation.
At present, however, the knowledge of fiber elongation and formation in plants from the viewpoint of molecular biology is very limited. Although many studies have been made on the elongation of plant cells, most of the control factors remain unknown and the control mechanisms have not yet been elucidated.
A cotton fiber is composed of a single cell that has been differentiated from an epidermal cell of the seed coat, and it develops through four stages, i.e., initiation, elongation, secondary cell wall thickening and maturation stages. More specifically, the elongation of a cotton fiber begins with that of an epidermal cell of the ovule just after flowering and the cotton fiber rapidly elongates and then completes elongation in about 25 days after the flowering. After that, the fiber elongation is stopped, and a secondary cell wall is formed and grown through maturation to become a mature cotton fiber.
Some reports have been made on the isolation of such genes associated with the elongation and formation of cotton fibers. John et al. describe the isolation of E6 gene that is expressed preferentially in the cotton fiber tissues on the 15th and 24th days after flowering (see John, M. E. and Crow, L. J., Proc. Natl. Acad. Sci. USA, 89, 5769–5773 (1992)) or H6 gene coding for a proline-rich protein that actively functions in the formation of secondary cell walls (see John, M. E. and Keller, G., Plant Physiol., 108, 669–676 (1995)). John further examined the effects of E6 gene on the cotton fiber characteristics by introduction of anti-sense E6 gene into cotton plants to reduce the expression level of endogenous E6 RNA (John, M. E., Plant Molecular Biology, 30, 297–306 (1996)). John, however, reported that although the expression level of E6 gene in fiber tissues is reduced, fiber length, fiber strength and fiber fineness are not significantly changed, and he concluded that E6 gene is not critical to the normal development of cotton fibers. Song et al. identified acyl carrier protein (ACP) cDNA from cotton plants by differential display method, which protein is specifically expressed in cotton fiber tissues (Song, P. and Allen, R. D., Biochimika et Biophysica Acta, 1351, 305–312 (1997)). As the gene associated with the cellulose synthesis in cotton fibers, cDNA coding for a catalytic subunit of cellulose synthase was isolated (Pear, J. R., Kawagoe, Y., et al., Proc. Natl. Acad. Sci. USA, 93, 12637–12642 (1996)). Kasukabe, one of the present inventors, and his coworkers have isolated and identified five genes from cotton plants by differential screening method and differential display method, which genes are specifically expressed at the cotton fiber elongation stage (assignees' own U.S. Pat. No. 5,880,100 and U.S. patent applications Ser. Nos. 08/580,545, 08/867,484 and 09/262,653). Some genes associated with the elongation and formation of cotton fibers have already been isolated from cotton plants; however, none have succeeded in modifying the fiber characteristics of cotton plants in practice.
The analysis of molecular mechanisms of plant cell wall construction led to the isolation of endoxyloglucan transferase (EXGT) as an enzyme that catalyzes molecular grafting between polysaccharide cross-links in the plant cell wall matrix (Nishitani, K. and Tominaga, R., J. Biol. Chem., 267, 21058–21064 (1992)). Xyloglucans are polysaccharides that cross-link individual cellulose microfibrils and play the main role in the net work structure of the cell wall involved in the cell elongation. For this reason, the transfer of xyloglucan cross-links by endoxyloglucan transferase is considered one of the important processes in the cell elongation. Some genes coding for endoxyloglucan transferase have been isolated from various plants including tomato (Lycopersicon esculentum) and mouse-ear cress (Arabidopsis thaliana) (Arrowsmith, D. A. and de Silva, J., Plant Mol. Biol., 28, 391–403 (1995); Okazawa, K., Sato, Y., et al., J. Biol. Chem., 268, 25364–25368 (1993)), and further from cotton plants (Gossypium spp.) (Shimizu, Y., Aotsuka, S., et al., Plant Cell Physiol., 38(3), 375–378 (1997)). There is, however, no report that endoxyloglucan transferase genes isolated from various plants or from cotton plants are actually introduced into cotton plants and examined for the effects on the cotton fiber characteristics.
Most of the plant catalase species are localized on the microbodies and they are detoxification enzymes that decompose metabolically produced toxic H2O2 into water and oxygen. Their functions in plants have not yet been fully elucidated, although some reports have been made that catalase is associated with the low temperature tolerance and pathogen resistance of plants (Sanchez-Casas, P. and Klessing, D. F., Plant Physiol., 106, 1675–1679 (1994); Prasad, T. K., Anderson, M. D., et al., Plant Cell, 6, 65–74 (1994)). There is, however, no report on the relationship between catalase and fiber characteristics of cotton plants.
Peroxidase is an enzyme that catalyzes the following oxidative reaction by hydrogen peroxide: H2O2+AH2→2H2O+A
Peroxidase genes have been isolated from various plants including tomato (Lycopersicon esculentum) and horseradish (Armoracia rusticana) (Roberts, E., Kolattukudy, P. E., Mol. Gen. Genet., 217, 223–232 (1987); Fujiyama, K., Tekemura, H., et al., Eur. J. Biochem., 173, 681–687 (1988)). Peroxidase is one of the cell wall enzymes and it exists through ionic bonding or covalent bonding in cell walls. One of the functions of peroxidase is the formation of lignin in the secondary components of cell walls. Peroxidase is considered to catalyze, in the presence of hydrogen peroxide, the reaction of forming dehydrogenation polymeric products from the lignin components (e.g., ferulic acid, p-coumaric acid) produced through shikimic acid pathway or cinnamic acid pathway (Gross, G. G. et al., Planta, 136, 271 (1977)). Another report describes that peroxidase is associated with the formation of intermolecular cross-links by the reaction of the respective tyrosine residues of two extensin molecules as the structural proteins of cell walls (Lamport, D. T. A., MSU-DOE Plant Research Laboratory 7th Annual Report, p. 65 (1982)). With respect to the relationship between peroxidase and cotton fibers, Rao et al. examined the peroxidase activity during the development of cotton fibers and showed that the peroxidase activity is lower in the cotton fiber elongation but significantly higher in the secondary wall thickening (Rao, N. R., Naithani, S. C., et al., Z. Pflanzenphysiol. Bd., 106, 157–165 (1982)). These results suggest a possibility that peroxidase is involved in the rigidifying of cotton fiber cell walls. John et al. confirmed that fiber strength can be increased by introduction of a peroxidase gene into cotton plants and over-expression of the gene in the cotton fiber tissues (WO95/-08914); however, they failed to obtain significant results on the fiber length or micronaire (i.e., fiber fineness).
As described above, some genes associated with the elongation and formation of cotton fibers have already been isolated; however, it seems to be the most important to actually introduce these genes into cotton plants to make sure of their effects on the level of practical use.